The hormone-related estrogen receptor (ER)alpha signaling has been intensively studied in breast cancer cells. Our recent study has implicated, for the first time, the epigenetic influence (i.e., chromatin remodeling and DMA methylation) on the transcription of ERalpha downstream target genes and provides a new direction of research in this classical signaling pathway. We hypothesize that disruption of normal ERalpha signaling in cancer cells may lead to long-term silencing of some downstream targets that are governed by epigenetic mechanisms. In this project, we will use computational and microarray-based approaches to define the molecular sequences leading to this epigenetic establishment. A positional weight matrix approach will be used to align and identify approximately 350 novel and known ERalpha targets, the 5'-end sequences of which will be arrayed for triple microarray analysis. In this microarray study, the exon-containing portions (exon 1) of these loci will be used for measuring expression levels of transcripts, their promoter sequences will be used for screening altered states of chromatin structure, and the GC-rich regions will be used for detecting changes of DNA methylation in the target sequences. Triple microarray will be used to simultaneously analyze these ERalpha targets in cultured breast cancer cells in which estrogen signaling is disrupted by small interference RNA. An empirical Bayesian approach will be used to model the sequences of epigenetic alterations after the RNA interference. Additional microarray experiments will also be conducted to reverse this epigenetic event by treating breast cancer cells with DNA demethylating agents and/or histone deacetylase inhibitors. The results may establish a model that upon ERalpha signal disruption, polycomb repressers and histone deacetylases are recruited to initiate transcriptional silencing in the interrogating ERalpha targets. This event is later accompanied by progressive accumulation of DNA methylation in the promoter regions of these targets, which leave a heritable mark that stably passes down to cells' progeny. Additional computation simulations will be used to explore other sequences of epigenetic events. The in vitro finding will be confirmed by conducting methylation microarray analysis in primary breast tumors. This proposed study is expected to give new information that hormonal insensitivity in breast cancer is, in part, attributed to epigenetically mediated silencing of multiple ERalpha targets and hope to provide a rationale for epigenetic therapies in breast cancer.